We investigated the effects of tyrosine kinase (TK) and nitric oxide synthase (NOS) inhibition on insulin-induced dilation of arterioles. We determined the arteriolar diameter, red blood cell velocity (V<sub>RBC</sub>) and blood flow changes in hamster cheek pouch microcirculation as affected by insulin in presence of TK and NOS inhibitors, genistein, piceatannol and N<sup>G</sup>-monomethyl- L-arginine ( L-NMMA). Microvessels were visualized by a fluorescent microscopy technique. Arteriolar diameter and V<sub>RBC</sub> were measured after topical application of insulin and genistein or piceatannol or L-NMMA. Insulin (10 µU/ml) induced diameter and V<sub>RBC</sub> increase in A3 and A2 arterioles by 30 ± 5 and 123 ± 4%, 16 ± 4 and 102 ± 3%, as percent of baseline values, respectively. After genistein or piceatannol prior to insulin A3 and A2 arterioles dilated by 10 ± 4, 5 ± 2% and 9 ± 4, 2 ± 1%, respectively. After L-NMMA prior to insulin A2 and A3, arteriole diameters increased by 12 ± 3 and 7 ± 2%, respectively. V<sub>RBC</sub> increased significantly in all the cases. TK and NOS inhibitors applied together abolished insulin-induced dilation with a reduction in V<sub>RBC</sub> and blood flow. In conclusion, full insulin-induced dilation of hamster cheek pouch arterioles requires TK signaling pathways. Furthermore, activation of insulin receptors, as well as other TK receptors, appears to be required for vasomotor tone regulation.